阅读说明：本技术 一种具有提高性功能功效的牡蛎肽及其制备方法 (Oyster peptide with sexual function improving effect and preparation method thereof ) 是由 蔡木易 谷瑞增 张海欣 马勇 魏颖 方磊 潘兴昌 董哲 陆路 周明 王雨辰 凌 于 2019-10-29 设计创作，主要内容包括：本发明提供一种具有提高性功能功效的牡蛎肽及其制备方法,所述牡蛎肽组成中至少包括肽段RI、IR以及VR；基于牡蛎肽的质量,所述RI的含量≥3.60mg/100g、IR的含量≥7.60mg/100g、VR的含量≥6.50mg/100g。本发明含有特定质量含量的肽段RI、IR以及VR的牡蛎肽在促进睾酮、二氢睾酮分泌等方面能表现出良好功效。(The invention provides oyster peptide with the effect of improving sexual function and a preparation method thereof, wherein the oyster peptide at least comprises peptide fragments RI, IR and VR; based on the mass of the oyster peptide, the RI content is more than or equal to 3.60mg/100g, the IR content is more than or equal to 7.60mg/100g, and the VR content is more than or equal to 6.50mg/100 g. The oyster peptide containing peptide segments RI, IR and VR with specific mass contents can show good effects on promoting the secretion of testosterone and dihydrotestosterone.)

1. The oyster peptide is characterized in that the oyster peptide at least comprises peptide fragments RI, IR and VR;

based on the mass of the oyster peptide, the RI content is more than or equal to 3.60mg/100g, the IR content is more than or equal to 7.60mg/100g, and the VR content is more than or equal to 6.50mg/100 g.

2. The oyster peptide according to claim 1, wherein the oyster peptide comprises peptides having a molecular weight of less than 1000u in an amount of 90% by mass or more.

3. The oyster peptide according to claim 1 or 2, wherein the oyster peptide is obtained by sequentially subjecting an oyster meat raw material to acid treatment to remove fat and polysaccharide, alkali treatment to denature protein, and enzymatic hydrolysis and purification treatment using neutral protease and papain.

4. A method for preparing the oyster peptide according to any one of claims 1 to 3, comprising the steps of:

1) adding water into the raw material of oyster meat to prepare mixed feed liquid, adding concentrated hydrochloric acid into the mixed feed liquid, stirring, and collecting precipitate after solid-liquid separation;

2) adding water to the precipitate to obtain slurry, adding alkali, and treating at 85-90 deg.C to denature protein to obtain denatured Concha Ostreae protein solution;

3) adding neutral protease and papain into the modified oyster protein solution for enzymolysis for 3-6h, and inactivating enzyme to obtain enzymolysis solution;

4) and centrifuging the enzymolysis liquid, and sequentially filtering and carrying out column chromatography treatment on the centrifuged supernatant to obtain the oyster peptide.

5. The method for preparing oyster peptide according to claim 4, wherein in the step 1), the mass-to-volume ratio of the oyster meat raw material to water is 1: (5-8), and adding 3-5mL of concentrated hydrochloric acid into each kilogram of the oyster meat raw material to perform acid treatment.

6. The method for producing oyster peptide according to claim 4, wherein the protein denaturation treatment uses solid sodium hydroxide, and the slurry is heated to 85 to 90 ℃ with stirring and kept at the temperature for 60 to 120min after the solid sodium hydroxide is added in a mass ratio of 0.8 to 1.0g of solid sodium hydroxide per kg of raw oyster meat.

7. The method for preparing oyster peptide according to claim 4, wherein the amount of the neutral protease is 0.8-1.6AU/1000g and the amount of the papain is 100000-300000U/1000g, based on the mass of the oyster meat material.

8. The method for preparing oyster peptide according to claim 4, wherein the filtering comprises: and filtering the centrifugal supernatant by using a ceramic membrane with the aperture of 50-200nm, and collecting filtrate.

9. The method for preparing oyster peptide according to claim 4 or 8, wherein the column chromatography comprises: and purifying the filtered filtrate by using a cation chromatographic column and a hydrophobic chromatographic column in sequence.

10. Use of the oyster peptide according to any one of claims 1 to 3 in a product for improving sexual function.

Technical Field

The invention relates to an oyster peptide with a sexual function improving effect and a preparation method thereof, belonging to the technical field of biology.

Background

Oysters, also known as fresh oysters, grow in warm and tropical seas and belong to bivalve soft body animals of the oyster family. The chicken has tender meat, outstanding delicate flavor and unique taste, and gradually becomes a favorite dining table food.

Researches show that the oysters have the characteristics of high protein content and low fat content besides good taste, contain 8 amino acids required by human bodies, and also contain glycogen, taurine, cystine, vitamin A, vitamin B1, vitamin B2, vitamin D, vitamin E, fucose, copper, zinc, manganese, barium, phosphorus, calcium, magnesium, aluminum, organic matters and the like, so that in recent years, researches on medicines or health-care products by taking the oysters as raw materials are also endless.

At present, in order to further improve the absorption of human body to the nutrient components in oysters, the oysters are mostly used as raw materials for enzymolysis, so that different small peptides which are beneficial to the life activities of living organisms or have physiological effects are separated. The small peptides have certain human metabolism and physiological regulation functions, can be directly absorbed in intestinal tracts, and have a quicker absorption speed than that of directly eating oysters, so that the oyster peptides generated by enzymolysis of the oysters are a new direction for deep processing of the oysters.

At present, during the production process of oyster peptide, more nutrition is provided for human bodies, the oysters are subjected to enzymolysis in the directions of resisting fatigue and improving immunity, so that peptide segments with other functions are inevitably lost during the enzymolysis process, the application range of the oysters is narrowed, and the further development of deep processing of the oysters is influenced.

Disclosure of Invention

The present invention provides an oyster peptide which exhibits excellent effects in promoting the secretion of testosterone, dihydrotestosterone, etc. by including specific functional peptide fragments, isoleucyl arginine (Ile-Arg, IR), arginyl isoleucine (Arg-Ile, RI), and valyl arginine (Val-Arg, VR) in a certain mass content therein.

The invention also provides a preparation method of the oyster peptide, which is characterized in that the oyster meat raw material is subjected to enzymolysis and separation and purification processes, so that the product definitely contains functional peptide fragments such as isoleucyl arginine (Ile-Arg, IR), arginyl isoleucine (Arg-Ile, RI) and valyl arginine (Val-Arg, VR) with certain mass contents.

The invention also provides application of the oyster peptide in a product for improving sexual function.

The invention provides oyster peptide, which at least comprises peptide fragments of isoleucyl arginine (Ile-Arg, IR), arginyl isoleucine (Arg-Ile, RI) and valyl arginine (Val-Arg, VR);

specifically, based on the total mass (dry basis) of the oyster peptides, the mass content of RI is more than or equal to 3.60mg/100g, the mass content of IR is more than or equal to 7.60mg/100g, and the mass content of VR is more than or equal to 6.50mg/100 g.

In addition, the oyster peptide also has the characteristic of small average molecular weight and easy absorption, and specifically, the mass content of the peptide with the molecular weight of less than 1000u in the oyster peptide is more than or equal to 90%.

The oyster peptide is obtained by using shelled oyster meat as a raw material and sequentially carrying out protein enrichment treatment (acid treatment for removing fat and polysaccharide), protein denaturation treatment (alkali treatment), enzymolysis treatment (neutral protease and papain) and separation and purification treatment; wherein the enzymolysis time is controlled to be 3-6h in the enzymolysis treatment, and the enzymolysis temperature is determined by the optimal activity temperature of neutral protease and papain, and can be 45-55 ℃.

During specific preparation, firstly, the oyster meat is subjected to acid treatment, namely, components such as fat, polysaccharide and the like in the oyster meat are separated, so that the protein content of an enzymolysis substrate is improved, protein enrichment is realized, and the enzymolysis efficiency is improved; then protein denaturation treatment is carried out on the product after protein enrichment, oyster protein is denatured under the action of alkali at a certain temperature, the microscopic expression is that the space structure of the oyster protein is destroyed, more enzymolysis points of the oyster protein are exposed, and the improvement of enzymolysis efficiency is further facilitated; after the protein denaturation treatment is finished, taking the protein denaturation product as an enzymolysis substrate, carrying out enzymolysis by using neutral protease and papain, and carrying out enzyme deactivation on the enzymolysis product (for example, heating to 115-125 ℃ and keeping the temperature for 15s) to obtain an enzymolysis liquid.

In the enzymolysis treatment, the dosage of the enzyme needs to be controlled to ensure that the peptide fragments RI, IR and VR in the product have high quality content as much as possible. Based on the mass of the oyster meat raw material, 0.8-1.6AU neutral protease, 100000-300000U papain can be used for every 1000g oyster meat raw material.

And (4) carrying out separation and purification treatment on the enzymolysis liquid to obtain the oyster peptide.

The separation and purification treatment of the enzymolysis liquid mainly comprises centrifugation, filtration and column chromatography treatment.

Specifically, firstly, centrifuging the enzymatic hydrolysate, and collecting a centrifugal supernatant; then filtering the centrifugal supernatant to separate macromolecular substances, wherein, for example, a ceramic membrane with the pore diameter of 50-200nm can be adopted; finally, column chromatography treatment is carried out on the filtered filtrate by using a cation exchange chromatography column and a hydrophobic chromatography column in sequence, so that peptide fragments RI, IR and VR in the enzymolysis liquid are reserved. In a specific embodiment, the cation exchange chromatography column can adopt 732 type cation exchange resin as a filler, and the particle size is between 0.315 and 1.25 mm; the packing material of the hydrophobic chromatographic column adopts Octyl sepharose 4FF type hydrophobic medium, and the particle size is between 45 and 165 mu m.

Subsequently, the liquid product collected by the column chromatography is concentrated and dried to obtain the desired oyster peptide, wherein at least the peptide fragments RI, IR and VR are contained.

The research shows that: the oyster peptide containing RI, IR and VR peptide segments with specific mass contents can remarkably promote the generation of testosterone and dihydrotestosterone, and is helpful for improving sexual function; in addition, the oyster peptide has the components with the molecular weight less than 1000u accounting for more than 90 percent, so that the oyster peptide is completely absorbed by intestinal tracts of human bodies and can more easily play a role in the human bodies.

The invention also provides a preparation method of the oyster peptide, which comprises the following steps:

1) adding water into the raw material of oyster meat to form mixed feed liquid, adding concentrated hydrochloric acid into the mixed feed liquid, stirring, centrifuging, and collecting precipitate;

2) adding water to the precipitate to obtain slurry, adding alkali, and treating at 85-90 deg.C to denature protein to obtain denatured Concha Ostreae protein solution;

3) adding neutral protease and papain into the modified oyster protein solution for enzymolysis for 3-6h, and inactivating enzyme to obtain enzymolysis solution;

4) and centrifuging the enzymolysis liquid, and sequentially filtering and carrying out column chromatography on the centrifuged supernatant to obtain the oyster peptide.

The invention does not limit the variety and the producing area of the oyster, and the raw material of the oyster meat can be fresh oyster meat after being peeled or unfrozen. After the oyster meat raw material is cleaned, in order to enable the subsequent protein enrichment treatment and protein denaturation treatment to have better effects, the cleaned oyster meat can be ground by a meat grinder.

Further, the mass volume ratio of the oyster meat raw material to water in the step 1) is 1: and (5-8) namely: mixing 1kg of Carnis Ostreae raw material with 5-8L of water to prepare mixed liquid. Subsequently, the temperature is controlled at 20-30 ℃, concentrated hydrochloric acid is added into the mixed solution and stirred for 60-120min, and the precipitate is collected by centrifugation (3000 and 4000rpm, 10 min). The aim of the operation is to separate components such as fat, polysaccharide molecules and the like in the oyster meat from protein and leave the components in the centrifugal supernatant, so that the protein in the oyster meat is enriched in the precipitate, and the subsequent enzymolysis treatment efficiency is improved. Generally, concentrated hydrochloric acid is used for treatment, and the dosage of the concentrated hydrochloric acid is calculated by oyster meat raw materials, and specifically, 3-5mL of concentrated hydrochloric acid is added into each kilogram of oyster meat raw materials from the aspects of operation and safety and convenience of products.

Collecting the precipitate in the step 1), adding water into the precipitate, and stirring to prepare slurry. Wherein the amount of water is calculated by oyster meat raw material, and 0.5-1L water is added into the precipitate per 1kg oyster meat raw material, and stirring to obtain slurry. The treatment can add proper amount of water into the protein-enriched precipitate to prepare slurry with certain fluidity, and is favorable for subsequent denaturation treatment and enzymolysis. When the water is added too little, the slurry has poor fluidity, is not beneficial to the action of an enzyme preparation, and is easy to cause the reduction of the enzymolysis efficiency; when the amount of water added is too large, the reaction volume is too large, and the load of subsequent treatment (such as concentration) is increased, which may also cause changes in the composition and structure of the product, and the treatment cost is also increased accordingly. Wherein, the water can be pure water, distilled water, deionized water, etc. Distilled water may be used in the preparation of the mixed liquid and slurry of the present invention.

Further, the protein denaturation treatment in step 2) is performed in an alkaline high-temperature environment, and an alkaline substance, generally a strong base, usually sodium hydroxide or potassium hydroxide, may be added to the prepared slurry, and a solid base may be directly added to the slurry. Specifically, adding solid sodium hydroxide into the slurry, keeping the temperature at 85-90 ℃ and continuously stirring for 60-120min, wherein 0.8-1.0g of solid sodium hydroxide is added into each kilogram of the oyster meat raw material. The condition can inactivate protease naturally existing in the oyster raw materials, avoids the influence on the enzymolysis effect of neutral protease and papain, can also destroy the space structure of oyster protein, exposes more enzyme digestion sites, and is easy to be subjected to protease enzymolysis. In addition, the slurry has high protein content, and moderate hydrolysis in an alkaline environment is favorable for solving the problems of poor slurry fluidity and viscous solution, and is favorable for subsequent enzymolysis.

The inventor conducts a great deal of research and investigation on how to enable the enzymolysis products of the oyster meat to contain RI, IR and VR peptide fragments with expected mass contents, and proves that the selection of the enzyme preparation and the corresponding separation process have a key influence on the result. The inventor unexpectedly finds that only neutral protease and papain are used for enzymolysis at the same time, which is beneficial to simultaneously obtaining RI, IR and VR peptide fragments and is beneficial to subsequent separation and purification of the RI, IR and VR peptide fragments, thereby further ensuring that the mass content of RI is more than or equal to 3.60mg/100g, the mass content of IR is more than or equal to 7.60mg/100g and the mass content of VR is more than or equal to 6.50mg/100 g.

In particular, in the enzymolysis of the invention, the amount of the neutral protease is 0.8-1.6AU/1000g and the amount of the papain is 100000-300000U/1000g based on the mass of the oyster meat raw material, namely, the neutral protease of 0.8-1.6AU and the papain of 100000-300000U are required for each kilogram of oyster meat raw material. The enzymolysis is carried out at the optimal activity temperature of neutral protease and papain, such as 45-55 ℃, the enzymolysis time is controlled to be 3-6h, the enzymolysis time is too short (< 1h) to be beneficial to the degradation of protein, and the time is too long (such as more than 7h) to cause the further degradation of the target peptide fragment. The enzymatic hydrolysis may also facilitate the formation of components of smaller molecular weight (e.g., peptides having a molecular weight of less than 1000 u) for absorption by the human body.

After the enzymatic hydrolysis is completed, the enzyme deactivation can be performed by using the conventional enzyme deactivation means in the art, for example, heating to 115-125 ℃ and maintaining for about 15 s.

Further, the rotation speed of the centrifugation in the step 4) can be controlled to be 3000-. After the centrifugation is finished, collecting the centrifugal supernatant and filtering the centrifugal supernatant by adopting a ceramic membrane with the aperture of 50-200 nm. The filtering can further screen out macromolecular proteins in the enzymolysis liquid, retain RI, IR and VR peptide fragments in the enzymolysis liquid and improve the mass content of the RI, IR and VR peptide fragments.

In the present invention, the filtered filtrate may be subjected to column chromatography. The column chromatography treatment comprises treating the filtrate with cation exchange chromatography column and hydrophobic chromatography column in sequence. Specifically, the treatment with a cation exchange chromatography column comprises: passing the filtrate through cation exchange chromatography column at linear velocity of 1-5cm/min, washing cation chromatography column with distilled water for 1-3CV, washing with 200mmol/L sodium chloride solution for 1-3CV, eluting with 700mmol/L sodium chloride solution, and collecting cation eluate for 2-4 CV; subsequently, the above cation eluent is passed through a hydrophobic chromatography column at a linear velocity of 1-5cm/min, the hydrophobic chromatography column is washed with 700mmol/L sodium chloride solution for 1-3CV to remove the non-adsorbed foreign proteins, and finally the hydrophobic chromatography column is eluted with distilled water for 1-3CV and collected.

The cation exchange chromatographic column can be balanced by distilled water after being regenerated, and the filler of the cation exchange chromatographic column can adopt 732 type cation exchange resin, and the particle size of the filler is between 0.315 and 1.25 mm; the hydrophobic chromatographic column is balanced by 700mmol/L sodium chloride solution, and the packing of the hydrophobic chromatographic column adopts Octyl sepharose 4FF type hydrophobic medium, and the particle size is 45-165 mu m.

Further, the eluent collected from the hydrophobic chromatographic column can be concentrated, for example, a rotary evaporator can be used for evaporation concentration, the vapor pressure during evaporation can be controlled to be 0.02-0.04MPa, the evaporation temperature is 60-80 ℃, and the concentration is stopped when the solid content in the concentrated solution is 10-20%, the solid content is convenient for subsequent drying treatment, specifically, when the solid content is too high, the viscosity of the system is increased, and the drying is not facilitated; when the solid content is too low, the drying energy consumption is increased, and the drying time is prolonged. Further, after concentration, drying may be performed to prepare the oyster peptide, and the drying may be, for example, freeze drying, and the freeze drying process may be: pre-freezing at-50 deg.C for 4-6 hr, vacuumizing, heating to 20-30 deg.C after the vacuum degree is lower than 20, and maintaining for 15-30 hr.

Through the enzymolysis process and the separation and purification process, not only can RI, IR and VR peptide fragments be obtained, but also the mass content of the peptide fragment RI is more than or equal to 3.60mg/100g, the mass content of the IR is more than or equal to 7.60mg/100g and the mass content of the VR is more than or equal to 6.50mg/100g through proper process parameters.

The invention also provides the application of the oyster peptide in products for improving sexual function, wherein the products include but are not limited to foods, health-care products and medicines.

A large amount of research data prove that the oyster peptide containing RI, IR and VR functional peptide segments with specific mass contents has remarkable capacity in promoting the generation of testosterone and dihydrotestosterone, and the oyster peptide provided by the invention has remarkable capacity of improving sexual functions, can be used for improving sexual function products and the like besides the conventional health application, thereby widening the application range of oysters and providing a new direction for deep processing of oysters.

The implementation of the invention has at least the following advantages:

1. the oyster peptide provided by the invention definitely contains RI, IR and VR functional peptide fragments, the mass content of RI is more than or equal to 3.60mg/100g, the mass content of IR is more than or equal to 7.60mg/100g, and the mass content of VR is more than or equal to 6.50mg/100g, so that the oyster peptide has the effect of remarkably improving sexual functions, is used as a raw material of related functional products, and provides a wider application prospect for oyster peptide products.

2. According to the preparation method of the oyster peptide, the oyster deep processing product with specific mass content of peptide fragments RI, IR and VR is obtained by adopting special pretreatment, enzymolysis, separation and purification processes.

Drawings

FIG. 1 is a gel chromatogram of the molecular weight distribution of oyster peptide in example 1 of the present invention;

FIG. 2 is a mass spectrum of a 1. mu.g/mL standard used in the identification of RI, IR and VR in the examples and comparative examples of the present invention;

FIG. 3 is a mass spectrum of RI, IR and VR in oyster peptide of example 1 at 3mg/mL in accordance with the present invention;

FIG. 4 is a gel chromatogram of the molecular weight distribution of oyster peptide in example 2 of the present invention;

FIG. 5 is a mass spectrum of RI, IR and VR in oyster peptide of example 2 at 3mg/mL in accordance with the present invention;

FIG. 6 is a gel chromatogram of the molecular weight distribution of oyster peptide in example 3 of the present invention;

FIG. 7 is a mass spectrum of RI, IR and VR in oyster peptide of example 3 at 3mg/mL in accordance with the present invention;

FIG. 8 is a mass spectrum of RI, IR and VR of 3mg/mL oyster peptide of comparative example 1 according to the present invention;

FIG. 9 is a mass spectrum of RI, IR and VR in 3mg/mL of the oyster peptide of comparative example 2 according to the present invention;

FIG. 10 is a mass spectrum of RI, IR and VR in 3mg/mL of the oyster peptide of comparative example 3 according to the present invention;

FIG. 11 is a mass spectrum of RI, IR and VR in the oyster peptide of comparative example 4 at 3mg/mL according to the present invention;

FIG. 12 is a graph showing the relationship between oyster peptide and the survival rate of TM3 cells at different mass concentrations in example 1 of the present invention;

FIG. 13 is a graph showing the relationship between the survival rate of TM3 cells in each test group;

FIG. 14 is a graph of the relationship between oyster peptide and the content of testosterone in TM3 cells at different mass concentrations in example 1 of the present invention;

FIG. 15 is a graph of the relationship between each test group and the amount of testosterone secreted by TM3 cells;

FIG. 16 is a graph showing the relationship between oyster peptide and the content of dihydrotestosterone in TM3 cells at different mass concentrations in example 1 of the present invention;

FIG. 17 is a graph of the relationship between the levels of dihydrotestosterone secreted by TM3 cells for each test group;

FIG. 18 is a graph showing the relationship between oyster peptide and NO content in TM3 cells at different mass concentrations in example 1 of the present invention;

FIG. 19 is a graph showing the relationship between each test group and the amount of NO in TM3 cells;

FIG. 20 is a graph showing the relationship between oyster peptide and SOD activity in TM3 cells at different mass concentrations in example 1 of the present invention;

FIG. 21 is a graph showing the relationship between SOD activity in TM3 cells in each test group;

FIG. 22 is a graph showing the relationship between oyster peptide and cGMP content in TM3 cells at different mass concentrations in example 1 of the present invention;

FIG. 23 is a graph showing the relationship between each test group and the intracellular cGMP content of TM 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

In the following examples and comparative examples, the neutral protease was purchased from Novoxil, 0.8 AU/g; papain was purchased from Pengbo of Nanning, 100 ten thousand U/g; alkaline protease was purchased from Novoxin, 2.4 AU/g; acid protease was purchased from Danisc, 2000 SAPU/g.

Example 1

The oyster peptide of this example was prepared as follows:

1. taking one kilogram of shelled oyster meat, unfreezing, mincing by a meat mincer, and adding 5L of distilled water to prepare a mixed solution; placing the mixed solution into a water bath with the temperature of 20 ℃ for stirring, adding 5mL of concentrated hydrochloric acid, continuously stirring for 60min, centrifuging for 10min at the rotating speed of 3500rpm by using a desktop centrifuge, and collecting precipitates;

2. adding 1L of distilled water into the precipitate, stirring to obtain slurry, adding 0.8g of sodium hydroxide solid into the slurry, heating to 90 deg.C under stirring, and maintaining for 60min to obtain modified Concha Ostreae protein solution;

3. cooling the modified oyster protein solution to 50 ℃ by a heat exchanger, adding 1.0g of neutral protease and 0.2g of papain, carrying out enzymolysis for 4h, and then carrying out UHT enzyme deactivation to obtain an enzymolysis solution;

4. centrifuging the enzymatic hydrolysate for 10min at 3500rpm with a desktop centrifuge, collecting supernatant, filtering with 200nm ceramic membrane, and collecting filtrate; passing the filtrate through cation exchange chromatography column (column model: xk16-50, diameter 16mm, height 400 mm; filler is 732 type cation exchange resin, and the filler particle diameter is 0.315-1.25 mm) at linear flow rate of 1cm/min, washing cation exchange chromatography column with distilled water at the same flow rate for 60min, washing with 200mmol/L sodium chloride solution for 60min, eluting with 700mmol/L sodium chloride solution, and collecting eluate 400 mL; passing the cation chromatography eluate through hydrophobic chromatography column (column model: xk16-50, diameter 16mm, height 400 mm; packing is Octyl sepharose 4FF type hydrophobic medium, and particle diameter is 45-165 μm) at linear flow rate of 1cm/min, eluting with 700mmol/L sodium chloride solution for 30min, eluting with 300mL hydrophobic chromatography column with distilled water, and collecting; concentrating the hydrophobic chromatography eluate to 100mL (Baume value: 17%) with a rotary evaporator, lyophilizing (-50 deg.C for 6 hr, vacuumizing, heating to 20 deg.C after vacuum degree is lower than 20, and maintaining for 20 hr) to obtain Concha Ostreae peptide powder 15 g.

Product assay

1. Molecular weight distribution detection of oyster peptides

The molecular weight detection is carried out by adopting the experimental method specified in the appendix of GB/T22492-2008 soybean peptide powder.

FIG. 1 is a gel chromatogram of the molecular weight distribution of oyster peptide in example 1 of the present invention.

Table 1 shows the molecular weight distribution data of the oyster peptide of example 1.

TABLE 1

2. And (3) detecting the contents of functional peptide fragments RI, IR and VR in the oyster peptide:

the peptide component in the oyster peptide in this example was identified using an ultra high performance liquid chromatograph Nexera X2 in combination with a triple quadrupole mass spectrometer system (shimadzu, japan).

Liquid chromatography conditions: a chromatographic column: inertsil ODS-3(5 μm,2.1 x 250 mm); mobile phase: a is 0.1% formic acid water solution, B is 0.1% formic acid acetonitrile solution; gradient elution procedure: 0-15min, B0-50%; 15-20min, B50-100%; 20-25min, B100%; 25.1-35min, B0%; flow rate: 0.2 mL/min; sample introduction volume: 1 mu L of the solution; column temperature: at 40 ℃.

Mass spectrum conditions: ionization mode: ESI, positive ion mode; ion spray voltage: +4.5 kV; flow rate of atomized gas: nitrogen gas is 3.0L/min; heating airflow rate: nitrogen gas is 10L/min; flow rate of drying gas: nitrogen gas 10L/min; DL temperature: 250 ℃; heating module temperature: 400 ℃; ion source temperature: 300 ℃; scanning mode: multiple Reaction Monitoring (MRM); residence time: 100 ms; delay time: 3 ms; MRM parameters: see table 2.

TABLE 2

Denotes quantitative ions

Preparing a peptide fragment standard substance: respectively and accurately weighing 20.0mg of RI, IR and VR standard substance powder, adding water to dissolve, uniformly mixing by vortex, and metering to 100mL, namely 200 mug/mL standard stock solution. Respectively taking 500 mu L of the standard stock solution, and fixing the volume to 10mL to obtain 10 mu g/mL of mixed standard mother solution. The above mixed standard mother liquor was diluted stepwise with pure water to a series of standard working solutions of 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5 and 10. mu.g/mL.

FIG. 2 is a mass spectrum of a 1. mu.g/mL standard sample used for identification of RI, IR and VR in examples and comparative examples of the invention, and FIG. 3 is a mass spectrum of RI, IR and VR in oyster peptide 3mg/mL in example 1 of the invention.

As can be seen from the comparison between fig. 3 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of example 1. According to detection, the RI content of the oyster peptide prepared in the example 1 is 3.68mg/100g, the IR content is 7.84mg/100g, and the VR content is 6.77mg/100 g.

Example 2

The oyster peptide of this example was prepared as follows:

1. taking five kilograms of shelled oyster meat, unfreezing, mincing by a meat mincer, and adding 25L of distilled water to prepare mixed liquid; stirring the mixed solution in a water bath at 20 ℃, adding 25mL of concentrated hydrochloric acid, continuously stirring for 60min, centrifuging for 10min at 3500rpm by using a desktop centrifuge, and collecting precipitate;

2. adding 5L of distilled water into the precipitate, stirring to obtain slurry, adding 5g of sodium hydroxide solid into the slurry, heating to 90 deg.C under stirring, and maintaining for 60min to obtain modified Concha Ostreae protein solution;

3. cooling the denatured oyster protein solution to 50 ℃ by a heat exchanger, adding 5g of neutral protease and 1g of papain, carrying out enzymolysis for 5h, and then carrying out UHT enzyme deactivation to obtain an enzymolysis solution;

4. centrifuging the enzymatic hydrolysate for 10min at 3500rpm with a desktop centrifuge, collecting supernatant, filtering with 200nm ceramic membrane, and collecting filtrate; passing the filtrate through cation exchange chromatography column (column model: xk26-100, diameter 26mm, height 60 mm; filler is 732 type cation exchange resin, and the filler particle diameter is 0.315-1.25 mm) at linear flow rate of 5cm/min, washing cation exchange chromatography column with distilled water at the same flow rate for 30min, washing with 200mmol/L sodium chloride solution for 30min, eluting with 700mmol/L sodium chloride solution, and collecting eluate 1500 mL; passing the cation chromatography eluate through hydrophobic chromatography column (column model: xk26-100, diameter 26mm, height 60 mm; packing is Octyl sepharose 4FF type hydrophobic medium, and particle diameter is 45-165 μm) at linear flow rate of 5cm/min, eluting with 700mmol/L sodium chloride solution for 30min, eluting with 1600mL hydrophobic chromatography column with distilled water, and collecting; concentrating the hydrophobic chromatography eluate to 400mL (Baume value: 18%) with a rotary evaporator, lyophilizing at (-50 deg.C for 6 hr, vacuumizing, heating to 20 deg.C after vacuum degree is lower than 20, and maintaining for 20 hr) to obtain Concha Ostreae peptide powder 70 g.

Product assay

1. The molecular weight distribution of the oyster peptide of this example was measured in the same manner as in example 1

FIG. 4 is a gel chromatogram of the molecular weight distribution of oyster peptide in example 2 of the present invention.

Table 3 shows the molecular weight distribution data of the oyster peptide of example 2.

TABLE 3

2. The content of functional peptide fragments RI, IR and VR in oyster peptide was determined by the same method as in example 1.

FIG. 5 is a mass spectrum of RI, IR and VR in oyster peptide of example 2 at 3mg/mL according to the present invention.

As can be seen from the comparison between fig. 5 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of example 2. According to detection, the RI content of the oyster peptide prepared in the example 2 is 3.75mg/100g, the IR content is 7.69mg/100g, and the VR content is 6.87mg/100 g.

Example 3

1. Taking 10kg of shelled oyster meat, unfreezing, mincing by a meat mincer, and adding 50L of distilled water to prepare a mixed solution; placing the mixed solution into a water bath at 25 ℃ for stirring, adding 50mL of concentrated hydrochloric acid, continuously stirring for 60min, centrifuging for 10min at 3500rpm by using a desktop centrifuge, and collecting precipitate;

2. adding 10L of distilled water into the precipitate, mixing, stirring to obtain slurry, adding 10g of sodium hydroxide solid into the slurry, heating to 90 deg.C under stirring, and maintaining for 90min to obtain modified Concha Ostreae protein solution;

3. cooling the denatured oyster protein solution to 50 ℃ by a heat exchanger, adding 12g of neutral protease and 2g of papain, carrying out enzymolysis for 5h, and then carrying out UHT enzyme deactivation to obtain an enzymolysis solution;

4. centrifuging the enzymatic hydrolysate for 10min at 3500rpm with a desktop centrifuge, collecting supernatant, filtering with 200nm ceramic membrane, and collecting filtrate; enabling the filtrate to pass through a cation exchange chromatography column at a linear flow rate of 1cm/min, washing the cation exchange chromatography column with distilled water at the same flow rate for 60min after the sample is loaded, washing with a 200mmol/L sodium chloride solution for 60min, eluting with a 700mmol/L sodium chloride solution, and collecting 3000mL of eluent; passing the cation chromatography eluate through hydrophobic chromatography column at linear flow rate of 1cm/min, eluting with 700mmol/L sodium chloride solution for 30min, eluting with distilled water for 300mL, and collecting; concentrating the hydrophobic chromatography eluate to 800mL (Baume value: 19%) with a rotary evaporator, lyophilizing (-50 deg.C for 6 hr, vacuumizing, heating to 20 deg.C after the vacuum degree is lower than 20, and maintaining for 20 hr) to obtain Concha Ostreae peptide powder 150 g. Wherein the type of the chromatographic column is XK 50/400.

Product assay

1. The molecular weight distribution of the oyster peptide of this example was measured by the same method as in example 1. fig. 6 is a gel chromatogram of the molecular weight distribution of the oyster peptide of example 3 of the present invention.

Table 4 shows the molecular weight distribution data of the oyster peptide of example 3.

TABLE 4

2. The content of functional peptide fragments RI, IR and VR in oyster peptide was determined by the same method as in example 1.

FIG. 7 is a mass spectrum of RI, IR and VR in oyster peptide of example 3 at 3mg/mL in accordance with the present invention.

As can be seen from the comparison between fig. 7 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of example 3. According to detection, the RI content of the oyster peptide prepared in the example 3 is 3.78mg/100g, the IR content is 7.86mg/100g, and the VR content is 6.63mg/100 g.

Comparative example 1

The preparation of this comparative example is essentially the same as that in example 2, with the only difference that: in the comparative example, after the centrifugal supernatant was filtered by using a 200nm ceramic membrane, the filtrate was directly concentrated to 800mL by a rotary evaporator (without being subjected to treatment by a cation exchange chromatography column and a hydrophobic chromatography column), and lyophilized to obtain 160g of oyster peptide powder.

Product assay

1. The content of functional peptide fragments RI, IR and VR in oyster peptide was determined by the same method as in example 1.

FIG. 8 is a mass spectrum of RI, IR and VR of the inventive 3mg/mL oyster peptide of comparative example 1.

As can be seen from the comparison between fig. 8 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of comparative example 1. Through detection, the RI content of the oyster peptide prepared in the comparative example 1 is 1.13mg/100g, the IR content is 2.14mg/100g, and the VR content is 1.95mg/100 g.

Comparative example 2

The preparation of this comparative example is essentially the same as that in example 2, with the only difference that: in the comparative example, 1500mL of eluent flowing out of the cation exchange chromatography column is collected, the 1500mL of eluent is directly concentrated to 700mL by a rotary evaporator without being treated by a hydrophobic chromatography column, and the oyster peptide powder 140g is obtained after freeze-drying.

Product assay

1. The content of functional peptide fragments RI, IR and VR in oyster peptide was determined by the same method as in example 1.

FIG. 9 is a mass spectrum of RI, IR and VR in the oyster peptide of comparative example 2 at 3mg/mL of the present invention.

As can be seen from the comparison between fig. 9 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of comparative example 2. Through detection, the RI content of the oyster peptide prepared in the comparative example 2 is 1.85mg/100g, the IR content is 3.79mg/100g, and the VR content is 3.25mg/100 g.

Comparative example 3

The preparation of this comparative example is essentially the same as that in example 2, with the only difference that: 5g of alkaline protease (novacin, alcalase2.4L) and 5g of neutral protease (novacin, Neutrase0.8L) are added during enzymolysis, the enzymolysis time is 4h, and the rest of post-treatment is the same as that of example 2.

Product assay

1. The content of functional peptide fragments RI, IR and VR in oyster peptide was determined by the same method as in example 1.

FIG. 10 is a mass spectrum of RI, IR and VR in the oyster peptide of comparative example 3 at 3mg/mL of the present invention.

As can be seen from the comparison between fig. 10 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of comparative example 3. According to detection, the RI content of the oyster peptide prepared in the comparative example 3 is 0.87mg/100g, the IR content is 2.08mg/100g, and the VR content is 1.62mg/100 g.

Comparative example 4

The preparation of this comparative example is essentially the same as that in example 2, with the only difference that: after washing with water, the slurry was washed, and the mixture was subjected to enzymolysis with 5g of acidic protease (Danisc, FOODPRO PAL) and 1g of papain for 4 hours without adding NaOH, and the other post-treatments were the same as in example 2.

Product assay

1. The content of functional peptide fragments RI, IR and VR in oyster peptide was determined by the same method as in example 1.

FIG. 11 is a mass spectrum of RI, IR and VR in the oyster peptide of comparative example 4 at 3mg/mL in accordance with the present invention.

As can be seen from the comparison between fig. 11 and fig. 2, the peptides RI, IR and VR are present in the oyster peptide of comparative example 4. According to detection, the RI content of the oyster peptide prepared in the comparative example 4 is 0.75mg/100g, the IR content is 1.63mg/100g, and the VR content is 1.30mg/100 g.

The samples were evaluated for improved sexual function by the following method.

1. MTT method for detecting influence of oyster peptide on proliferation of testicular interstitial cells TM3

MTT operation flow: cell density was diluted to 1X 10⁵Cells per mL were added to 96-well plates at 100 μ L/well. After the cells are observed to be uniformly attached to the wall in 24 hours, discarding supernatant, adding PBS (phosphate buffer solution) to wash the cells for 2-3 times, treating the cells with cell culture solution containing oyster peptides, setting the holes without samples as normal control groups, setting 4 more holes in the same 96-hole plate for each concentration gradient, discarding cell supernatant after 24 hours, washing the cells for 2-3 times with PBS (phosphate buffer solution), then adding 100 mu L of MTT solution (0.5mg/mL) into each hole, continuing incubation for 4 hours at 37 ℃, carefully removing the culture solution and MTT, adding 100 mu L of DMSO solution into each hole, and shaking for 10 minutes to dissolve crystals. The absorbance was measured at 490nm using a microplate reader, and the cell viability was determined as OD/control OD × 100%.

2. Detection of testosterone

Culturing TM3 cells with oyster peptide culture solution, centrifuging the culture solution after 24h, taking supernatant, mixing uniformly, and detecting the content of testosterone according to the specification of an ELISA kit.

Testosterone detection operation flow: taking out the plate strips needed by the test from the aluminum foil bags which are balanced and sealed to room temperature, taking back the unused plate strips and drying agents into the aluminum foil bags to fasten the self-sealing strips, sealing the bags, and putting the bags into the room at 2-8 ℃. Adding the standard substance and the sample to be detected into all the holes on the enzyme label plate according to 50 mu L/hole, and setting 4 Blank holes. And thirdly, adding 50 mu L of enzyme-labeled antigen working solution into each hole (except Blank holes), adding 50 mu L of rabbit anti-testosterone antibody working solution again according to the same sample adding sequence, sealing the reaction holes by using a sealing plate membrane after uniformly mixing, and incubating for 1h at 37 ℃. Fourthly, the power supply of the microplate reader is turned on 20min in advance, the instrument is preheated, and the detection program is set. Fifthly, carefully uncovering the sealing plate film, washing for 3 times by using a plate washing machine, and finally drying as much as possible. Sixthly, mixing the chromogenic substrate A and the chromogenic substrate B in equal volume according to the actual using amount of the test, adding the chromogenic substrate A and the chromogenic substrate B into the holes according to the volume of 100 mu L/hole, sealing the reaction holes by using a sealing plate membrane, and incubating for 15min in a dark place at 37 ℃. Seventhly, adding 50 mu L of stop solution per hole, and uniformly mixing to obtain the OD₄₅₀Value (within 10 min).

3. Dihydrotestosterone (DHT) content

And (3) acting the oyster peptide culture solution on TM3 cells, centrifuging the culture solution after 24 hours, taking supernatant, mixing uniformly, and detecting the content of dihydrotestosterone according to the specification of an ELISA kit.

Dihydrotestosterone detection operation flow: standard wells were added at 50. mu.L/well. ② adding 40 mul of sample diluent into the sample hole, then adding 10 mul of sample to be measured, and adding nothing into the blank hole. Thirdly, the reaction hole is sealed by a sealing plate membrane and incubated for 45min at 37 ℃. Fourthly, carefully uncovering the sealing plate film, washing for 3 times by a plate washing machine, and finally drying as much as possible. Adding 50 μ L antibody into each hole except blank hole. Sixthly, sealing the reaction hole by using a sealing membrane, and incubating for 30min at 37 ℃. Seventhly, sealing the reaction hole by using a sealing plate film, and incubating at 37 ℃ for 30 min. The washing steps are the same as the fifth step. Ninthly, adding 50 mu L of developing solution A and 50 mu L of developing solution B into each hole, mixing the solution evenly and gently, and incubating the solution for 15min at 37 ℃ in a dark place. ② 0 adding stop solution 50 μ L/hole, mixing uniformly to measure OD₄₅₀The value is obtained.

4. Determination of the NO content

And (3) acting the oyster peptide culture solution on TM3 cells, centrifuging the culture solution after 24 hours, taking supernatant, mixing uniformly, and detecting the content of NO according to the instruction of an NO determination kit.

NO detection operation flow: preparing a reagent: all reagents were removed and equilibrated to room temperature for use. 100 μ M standard working solution: add 5. mu.L of concentrated standard into 495. mu.L of reaction buffer and mix well. Preparing total nitric oxide detection working solution: adding 1 volume of NO into 40 volumes of Griess Reagent I according to the quantity of samples₃A reducing agent and 40 volumes of Griess Reagent II to prepare a proper amount of reaction working solution, and the reaction working solution is fully mixed and used within 1 hour. Numbering and sample adding are carried out on the plate holes in a 96-hole plate: and sealing the cover plate film, placing the microporous plate at 60 ℃ for incubation for 10min, taking out the microporous plate after the time is up, placing the microporous plate into a 37 ℃ incubator for incubation for 60min, and then taking out the microporous plate and placing the microporous plate into a 540nm position in an enzyme-labeling instrument for reading. Quantitatively calculating the total nitrogen monoxide in the sample: sample total nitric oxide content (μ M) ═ sample well OD value-blank well OD value)/(standard well OD value-blank well OD value) × standard concentration (100 μ M) × n (dilution factor).

5. Determination of SOD (superoxide dismutase) content in mouse testicular interstitial cell TM3

The oyster peptide culture solution is acted on TM3 cells, the cells are cracked after 24 hours to obtain lysate, and the content of SOD is detected according to the SOD determination kit.

SOD detection operation process: collecting about 2X 10⁶Centrifuging the cells at 800g and 4 ℃ for 2min, then removing the supernatant, washing the cells with cold PBS, centrifuging, and then removing the supernatant; add 500. mu.L of pre-chilled lysate (50mM Potassium phosphate, 0.1mM EDTA, 0.5% Triton X-100) to resuspend the cells, stand on ice for 10min, centrifuge at 12000g for 5min at 4 ℃ and aspirate the supernatant for detection. Secondly, numbering and sample adding are carried out on the plate holes in a 96-pore plate, after the sample adding is finished, the micropore plate is placed at room temperature for incubation for 10min, and after the time, the micropore plate is taken out and placed in a 550nm position in an enzyme labeling instrument for reading. ③ the quantitative calculation of superoxide dismutase (SOD) in the sample: inhibition (%) - (control well OD value-sample well OD value)/control well OD value. The enzyme activity at 50% inhibition was defined as 1U, and the supernatant of cell lysate was ultra-highSuperoxide dismutase (SOD) content (U/mg) ═ inhibition rate/50%/protein concentration of sample to be tested (mg/mL) × 100 × n (dilution factor)

6. cGMP (cyclic guanosine monophosphate) assay

And (3) acting the oyster peptide culture solution on TM3 cells, cracking the cells after 24 hours to obtain lysate, and detecting the content of cGMP according to the specification of a cGMP determination kit.

cGMP detection operation flow: taking out the laths required by the test from the aluminum foil bags which are balanced and sealed to room temperature, taking back the unused laths and drying agents into the aluminum foil bags to fasten the self-sealing strips, sealing the bags, and putting the bags into the room at 2-8 ℃. ② adding a neutralization reagent into all the holes of the enzyme label plate according to 50 mu L per hole. Secondly, selecting proper holes and adding the standard substance and the sample to be detected according to 100 mu L/hole. ③ 50 mu LcGMP conjugate is added into each hole, and then 50 mu LcGMP ELISA antibody is added. Fourthly, after mixing evenly, the reaction holes are sealed by a sealing plate membrane, and the mixture is placed on a flat plate oscillator for 500prm incubation for 2 hours. Fifthly, carefully uncovering the sealing plate film, washing for 3 times by using a plate washing machine, and finally drying as much as possible. Sixthly, 200 mu L of pNpp substrate solution is added to each well, and the incubation is continued for 1h at room temperature. Seventhly, adding 50 mu L of stop solution into each hole, and uniformly mixing to obtain the OD₄₅₀Value (within 10 min).

The following tests were conducted using the oyster peptides of examples 1 to 3 and the oyster peptides of comparative examples 1 to 4 as samples.

Test example 1

a. In order to detect the influence of different concentrations of oyster peptide culture solutions on the activity of TM3 cells, oyster peptide culture solutions of example 1 with the concentration series of 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, 800. mu.g/mL, 1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL and 10mg/mL were set to act on TM3 cells, and the optimal acting concentration of oyster peptide was determined by the MTT method.

FIG. 12 is a graph showing the relationship between the oyster peptide and the survival rate of TM3 cells at different mass concentrations in example 1 of the present invention. As shown in fig. 12, when the concentration of oyster peptide was in the range of 100 μ g/mL to 1mg/mL, there was no significant effect on the survival rate of TM3 cells (P >0.05), and when the concentration of oyster peptide was in the range of 2mg/mL to 10mg/mL, there was an inhibitory effect on the activity of cells, and there was concentration dependence, showing that the oyster peptide had a toxic effect on cells at a higher concentration, as compared to the control group to which the oyster peptide was not added.

In order to ensure the normal growth of the cells in the subsequent experiment, the sample is loaded at a concentration below the nontoxic concentration. Therefore, the loading concentrations of the oyster peptides of example 1 were set to 100. mu.g/mL, 400. mu.g/mL, and 1 mg/mL.

b. The oyster peptides of examples 1 to 3 and comparative examples 1 to 4 were prepared into a culture solution at a concentration of 400. mu.g/mL, respectively, and the influence of the oyster peptides of examples 1, 2, 3 and comparative examples 1, 2, 3, 4 on the survival rate of TM3 cells was examined by the MTT method.

FIG. 13 is a graph showing the relationship between the survival rate of TM3 cells in each test group. As shown in fig. 13, the oyster peptides of examples 1-3 were able to provide higher survival rates of TM3 cells compared to the oyster peptides of comparative examples 1-4.

Test example 2

In this test example, TM3 cells were treated with different concentrations of the oyster peptide culture solution of example 1 and the same concentrations of the oyster peptide culture solutions of examples 1 to 3 and comparative examples 1 to 4 for 24 hours, and then the supernatant medium of the cells was collected and the content of testosterone secreted from the cells was measured by ELISA.

a. The oyster peptides of example 1 were prepared into culture solutions of different concentrations, respectively, and the influence of different concentrations of the oyster peptides of example 1 on the content of testosterone secreted by TM3 cells was examined by ELISA.

FIG. 14 is a graph of the relationship between oyster peptide and the content of testosterone in TM3 cells at different mass concentrations in example 1 of the present invention. As shown in fig. 14, the oyster peptides at different concentrations showed a significant increase in testosterone secretion (P <0.05) to TM3 cells and were concentration-dependent compared to the control group without any oyster peptides. When the concentration of the oyster peptide is 1mg/mL, the promotion effect on the secretion of testosterone by TM3 cells is higher than that of positive control sildenafil (200 mu g/mL), which indicates that the oyster peptide indeed has the effect of promoting the secretion of testosterone by TM3 cells.

Under the condition of ensuring that the total amount of the sample is constant, in order to examine the influence of different sample adding time on cells, the concentrations of 100 mu g/mL and 400 mu g/mL are divided into two times of sample adding (24h twice, once every 12 h), namely 100 mu g/mL is divided into two times, and each time is 50 mu g/mL; 400. mu.g/mL was divided into two portions of 200. mu.g/mL each. As can be seen from fig. 14, two equal aliquots of oyster peptide more promoted testosterone secretion by TM3 cells.

b. The oyster peptides of examples 1 to 3 and comparative examples 1 to 4 were prepared into a culture solution at a concentration of 400. mu.g/mL, respectively, and the influence of the oyster peptides of examples 1, 2, 3 and comparative examples 1, 2, 3, 4 on the content of testosterone secreted from TM3 cells was examined by ELISA.

FIG. 15 is a graph of the relationship between each test group and the amount of testosterone secreted by TM3 cells. As shown in fig. 15, the oyster peptides of examples 1-3 significantly promoted secretion of testosterone by TM3 cells compared to the oyster peptides of comparative examples 1-4, and the example 1 had the strongest effect of promoting testosterone production by TM3 cells.

Test example 3

Dihydrotestosterone is a steroid hormone secreted by the testes, is the main androgen in the human body, is related to the development of male secondary sex characteristics, and plays an important role in maintaining normal sexual desire. In this test example, TM3 cells were treated with different concentrations of the oyster peptide culture solution of example 1 and the same concentrations of the oyster peptide culture solutions of examples 1 to 3 and comparative examples 1 to 4 for 24 hours, and then the supernatant medium of the cells was collected and the content of dihydrotestosterone secreted from the cells was measured by ELISA.

a. The oyster peptides of example 1 were prepared into culture solutions of different concentrations, respectively, and the influence of the oyster peptides of example 1 of different concentrations on the content of dihydrotestosterone secreted by TM3 cells was examined by ELISA. FIG. 16 is a graph showing the relationship between oyster peptide and the content of dihydrotestosterone in TM3 cells at different mass concentrations in example 1 of the present invention. As shown in fig. 16, the content of dihydrotestosterone in TM3 cells of the control group was significantly lower than that in TM3 cells treated with oyster peptide, compared to the control group without any culture medium, indicating that oyster peptide has the effect of promoting and improving dihydrotestosterone secretion from leydig cells and has concentration dependence. The positive control sildenafil (200 mu g/mL) also has the effect of remarkably promoting the generation of testosterone by TM3 cells, and the promoting effect on the cells is similar to that of the positive control sildenafil when the concentration of the oyster peptide is 1 mg/mL.

Under the condition of ensuring that the total amount of the sample is constant, in order to examine the influence of different sample adding time on cells, the concentrations of 100 mu g/mL and 400 mu g/mL are divided into two times of sample adding (24h twice, once every 12 h), namely 100 mu g/mL is divided into two times, and each time is 50 mu g/mL; 400. mu.g/mL was divided into two portions of 200. mu.g/mL each. As can be seen in fig. 16, two equal aliquots of oyster peptide more promoted dihydrotestosterone secretion by TM3 cells.

b. The oyster peptides of examples 1 to 3 and comparative examples 1 to 4 were prepared into a culture solution at a concentration of 400. mu.g/mL, respectively, and the influence of the oyster peptides of examples 1, 2, 3 and comparative examples 1, 2, 3, 4 on the content of dihydrotestosterone secreted from TM3 cells was examined by ELISA.

FIG. 17 is a graph of the relationship between the levels of dihydrotestosterone secreted by TM3 cells in each test group. As shown in fig. 17, the oyster peptides of examples 1 to 3 significantly promoted dihydrotestosterone secretion from TM3 cells compared to the oyster peptides of comparative examples 1 to 4, and example 1 had the strongest promotion effect on dihydrotestosterone production from TM3 cells.

Test example 4

NO is a fat-soluble small molecule which is unstable in chemical property, is produced in vivo under the catalytic reaction of nitric oxide synthase, can cause the expansion of blood vessels in vivo, is an important messenger for the relaxation of the corpus cavernosum of the penis, and has a decisive role in the process of inducing and maintaining the erection of the penis. This test example used different concentrations of oyster peptide culture solution of example 1 and the same concentrations of oyster peptide culture solutions of examples 1-3 and comparative examples 1-4 to act on TM3 cells, respectively, after 24h, the culture solutions were centrifuged, the supernatants were collected and mixed, and the effect of oyster peptide on the NO content in TM3 cells was determined as indicated by the NO assay kit.

a. The oyster peptides of example 1 were prepared into culture solutions of different concentrations, respectively, and the influence of the oyster peptides of example 1 of different concentrations on the intracellular NO content of TM3 was examined by means of NO assay kits. FIG. 18 is a graph of oyster peptide in relation to NO content in TM3 cells at different mass concentrations in example 1 of the present invention. As shown in fig. 18, the NO content of TM3 cells was significantly increased in both the positive control sildenafil (200 μ g/mL) and the oyster peptide test group (p <0.05) compared to TM3 cells in the control group without any culture solution.

Under the condition of ensuring that the total amount of the sample is constant, in order to examine the influence of different sample adding time on cells, the concentrations of 100 mu g/mL and 400 mu g/mL are divided into two times of sample adding (24h twice, once every 12 h), namely 100 mu g/mL is divided into two times, and each time is 50 mu g/mL; 400. mu.g/mL was divided into two portions of 200. mu.g/mL each. As can be seen from fig. 18, when the oyster peptide was treated with TM3 cells in two separate applications, the increase in cellular NO content was more significant.

b. The oyster peptides of examples 1 to 3 and comparative examples 1 to 4 were prepared into a culture solution at a concentration of 400. mu.g/mL, respectively, and the influence of the oyster peptides of examples 1, 2, 3 and comparative examples 1, 2, 3, 4 on the intracellular NO content of TM3 was examined by means of an NO measurement kit.

FIG. 19 is a graph showing the relationship between each test group and the intracellular NO content of TM 3. As shown in fig. 19, the oyster peptides of examples 1 to 3 significantly promoted the NO content in TM3 cells, and example 1 had the strongest promotion effect on the NO content in TM3 cells, compared to the oyster peptides of comparative examples 1 to 4.

Test example 5

Superoxide dismutase (SOD) is an active substance in organisms, and can eliminate harmful substances generated in the metabolism process of organisms. SOD can catalyze the conversion of superoxide radicals into hydrogen peroxide and molecular oxygen, and plays a key role in resisting cell injury caused by oxygen radicals. In order to evaluate the oxidation environment of TM3 cells, different concentrations of the oyster peptide culture solution of example 1 and the oyster peptide culture solution of examples 1-3 and comparative examples 1-4 were applied to TM3 cells, respectively, and after 24 hours, the culture solutions were centrifuged, and the supernatant was collected and mixed, and the activity of SOD in the cells was measured according to the SOD measurement kit.

a. The oyster peptides of example 1 were prepared into culture solutions of different concentrations, and the effect of the oyster peptides of example 1 on the SOD enzyme activity in TM3 cells was examined by SOD enzyme assay kit. FIG. 20 is a graph showing the relationship between oyster peptide and SOD activity in TM3 cells at different mass concentrations in example 1 of the present invention. As shown in figure 20, the activity of SOD enzyme in oyster peptide group cells is remarkably improved (P is less than 0.05) compared with that of a control group without any culture solution, and the effect of a high-dose group (1mg/mL) is obviously better than that of a low-dose group (100 mu g/mL), and certain dose dependence is presented. After TM3 cells are treated by oyster peptide, the enzyme activity of SOD can be obviously enhanced, so that the oxidation environment in the cells is favorable for the generation of androgens such as testosterone.

Under the condition of ensuring that the total amount of the sample is constant, in order to examine the influence of different sample adding time on cells, the concentrations of 100 mu g/mL and 400 mu g/mL are divided into two times of sample adding (24h twice, once every 12 h), namely 100 mu g/mL is divided into two times, and each time is 50 mu g/mL; 400. mu.g/mL was divided into two portions of 200. mu.g/mL each. As can be seen from fig. 20, when the oyster peptide was treated with TM3 cells in two separate applications, the increase in cellular SOD activity was more significant.

b. The oyster peptides of examples 1 to 3 and comparative examples 1 to 4 were prepared into culture solutions at a concentration of 400. mu.g/mL, respectively, and the influence of the oyster peptides of examples 1, 2 and 3 and comparative examples 1, 2, 3 and 4 on the SOD activity in TM3 cells was examined by an SOD assay kit.

FIG. 21 is a graph showing the relationship between SOD activity in TM3 cells in each test group. As shown in fig. 21, the oyster peptides of examples 1 to 3 significantly promoted the activity of TM3 intracellular SOD compared to the oyster peptides of comparative examples 1 to 4, and example 1 had the strongest promotion effect on the activity of TM3 intracellular SOD.

Test example 6

In this test example, the oyster peptide culture solutions of example 1 and those of examples 1 to 3 and comparative examples 1 to 4 were applied to TM3 cells at different concentrations, respectively, and after 24 hours, the culture solutions were centrifuged, and the supernatants were collected and mixed, and the cGMP content in TM3 cells was measured according to the cGMP assay kit.

a. The oyster peptide of example 1 was prepared into culture solutions of different concentrations, and the effect of the oyster peptide of example 1 of different concentrations on the cGMP content in TM3 cells was examined by a cGMP assay kit. FIG. 22 is a graph showing the relationship between oyster peptide and cGMP content in TM3 cells at different mass concentrations in example 1 of the present invention. As shown in fig. 22, oyster peptide significantly increased cGMP content in TM3 cells, and the effect was stronger as the concentration of oyster peptide increased, compared to the control without any culture solution.

Under the condition of ensuring that the total amount of the sample is constant, in order to examine the influence of different sample adding time on cells, the concentrations of 100 mu g/mL and 400 mu g/mL are divided into two times of sample adding (24h twice, once every 12 h), namely 100 mu g/mL is divided into two times, and each time is 50 mu g/mL; 400. mu.g/mL was divided into two portions of 200. mu.g/mL each. As can be seen in fig. 22, we also found that loading an equal amount of oyster peptide twice more promoted cGMP secretion from TM3 cells.

b. The oyster peptides of examples 1 to 3 and comparative examples 1 to 4 were prepared into a culture solution at a concentration of 400. mu.g/mL, respectively, and the influence of the oyster peptides of examples 1, 2, 3 and comparative examples 1, 2, 3, 4 on the intracellular cGMP content of TM3 was examined by means of a cGMP assay kit.

FIG. 23 is a graph showing the relationship between each test group and the intracellular cGMP content of TM 3. As shown in fig. 23, the oyster peptides of examples 1 to 3 significantly promoted the increase of cGMP content in TM3 cells, and example 1 had the strongest promoting effect on cGMP secretion by TM3 cells, compared to the oyster peptides of comparative examples 1 to 4.

In the context of figures 12-23,^*"representative vs. blank, P<0.05。

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.